Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal

• Published in: Sheng wu yi xue gong cheng xue za zhi 2021-02, Vol.38 (1), p.111-121
• Main Authors: Gao, Mengyu, Zhu, Xinglong, Wang, Shisheng, Zhang, Bingqi, Zhang, Yunlin, He, Yuting, Zhou, Yanyan, Li, Shun, Yang, Guang, Liao, Guangneng, Bao, Ji, Bu, Hong
• Format: Article
• Language: Chinese
• Subjects: Animals; Bioreactors; Cell culture; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Cas Systems - genetics; DNA microarrays; FAH gene; Gene Editing; Genetic modification; Genomes; Hogs; Reproductive cycle; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; Screening; Swine; Technology utilization
• ISSN: 1001-5515
• DOI: 10.7507/1001-5515.202006032

The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained gene edited cells, laying a foundation for the subsequent production of knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.